Fluoroquinolone resistance does not facilitate phage Φ13 integration or excision in Staphylococcus aureus

Prophages of the ΦSa3int family are commonly found in human-associated strains of Staphylococcus aureus where they encode factors for evading the human innate immune system. In contrast, they are usually absent in livestock-associated methicillin-resistant S. aureus (LA-MRSA) strains where the phage attachment site is mutated compared to the human strains. However, ΦSa3int phages have been found in a subset of LA-MRSA strains belonging to clonal complex 398 (CC398), including a lineage that is widespread in pig farms in Northern Jutland, Denmark. This lineage contains amino acid changes in the DNA topoisomerase IV and the DNA gyrase encoded by grlA and gyrA, respectively, which have been associated with fluoroquinolone (FQ) resistance. As both of these enzymes are involved in DNA supercoiling, we speculated that the mutations might impact recombination between the ΦSa3int phage and the bacterial chromosome. To examine this, we introduced the FQ resistance mutations into S. aureus 8325-4attBLA that carry the mutated CC398-like bacterial attachment site for ΦSa3int phages. When monitoring phage integration and release of Φ13, a well-described representative of the ΦSa3int phage family, we did not observe any significant differences between the FQ-resistant mutant and the wild-type strain. Thus our results suggest that mutations in grlA and gyrA do not contribute to the presence of the ΦSa3int phages in LA-MRSA CC398.


INTRODUCTION
Staphylococcus aureus is an opportunistic bacterial pathogen that colonizes about one-third of the human population in addition to a wide range of animals [2]. Staphylococcal strains are divided into lineages with some being livestock-associated (LA) while others are hospital or community associated. Despite the preference of LA-strains for livestock, a significant fraction of human infections are caused by LA-strains that are resistant to methicillin (LA-MRSAs) and close contact with livestock is a risk factor [3,4].
Host specificity of S. aureus is tightly linked to its content of mobile genetic elements [5]. Most human strains carry prophages of the ΦSa3int family that are integrated in the bacterial attachment site (attB) located in the hlb gene [6]. ΦSa3int phages carry an immune evasion cluster (IEC) that encodes one or more immune evasion factors and their presence promotes human colonization and human-to-human transmission of S. aureus [7][8][9]. In contrast, LA-MRSA strains belonging to the clonal complex CC398 commonly lack ΦSa3int phages. This may be related to their variant phage attachment site (designated attB LA ) [7,10,11] where OPEN ACCESS the sequence 5′-TGTATCCGAATTGG-3′ differs from non-livestock, human strains at the underlined nucleotides [10,11]. These nucleotide changes decrease the integration frequency of ΦSa3int phages and lead to integration at other locations in the bacterial genome than the hlb gene [10][11][12][13][14]. Despite the reduced integration frequency there is an overrepresentation of strains with ΦSa3int phages in human isolates of CC398 compared to livestock isolates [8]. Also the presence of the prophage is associated with 'spillover' events where humans are infected with LA-MRSA CC398 strains without having had livestock contact, indicating an increased risk of human-to-human transmission [15,16]. This is especially problematic in areas with intensive pig farming where those strains have become a serious human disease burden [17,18].
We recently identified and characterized 20 LA-MRSA CC398 strains from Northern Jutland, Denmark with 17 isolates obtained from humans and three from pigs, that all harboured IEC-carrying ΦSa3int prophages [13]. The prophages could be divided into six variants on the basis of their phylogenetic relationship, IEC type, and chromosomal integration site and each variant was unique to a single household. Interestingly, all isolates exhibited decreased susceptibility to fluoroquinolones (FQ) and belonged to the same lineage, L1 (Fig. 1). As these findings indicated that there had been several integration events by ΦSa3int phages, we wondered if there may be a link between FQ resistance and ΦSa3int phage integration or stability.
FQs are a class of synthetic broad-spectrum antibiotics, and newer generations of this class are effective against a broad spectrum of Gram-negative and -positive bacteria, including S. aureus [19]. In humans they are frequently prescribed [20], whereas in livestock, FQs are tightly regulated and only prescribed after laboratory susceptibility testing [21]. FQs primarily target the A-subunits of the bacterial DNA topoisomerase IV and the DNA gyrase encoded by grlA and gyrA, respectively [22]. Both enzymes shape DNA topology through cleavage, entwinement and re-ligation of DNA double strands. Binding of FQ at the enzyme-DNA intersection leads to stalling of DNA re-ligation and synthesis, which ultimately results in cell death [23]. Presence (black) and absence (light grey) of the IEC and Sa3int phage as well as grlA and gyrA mutations conferring fluoroquinolone resistance is indicated. The tree was rooted according to [6] and the scale bar represents the number of nucleotide substitutions per variable site. FQ resistance is mediated by mutations clustered in the 5′-end of gyrA (between residues 68-107) and grlA (between residues 64-103) [24,25] leading to reduced binding of FQ at the catalytic sites. When S. aureus is exposed to FQ, mutations occur stepwise with grlA being modified first leading to a 64-fold increase in the minimal inhibitory concentration (MIC) to ciprofloxacin from 0.5 µg ml −1 to 32 µg ml −1 . In a subsequent step, mutations in gyrA confer high level resistance with an increase in MIC to 128 µg ml −1 [25].
The structural changes of DNA-modifying enzymes associated with FQ resistance may also affect DNA supercoiling. This has been seen in Campylobacter jejuni [ To examine whether there may be a link between FQ resistance in LA-MRSA CC398 strains and lysogeny of ΦSa3int phages, we introduced the gyrA/grlA mutations in S. aureus strain 8325-4attB LA , that is a derivative of 8325-4 carrying the CC398like bacterial attachment site (attB LA ) [11]. Subsequently we compared lysogenization frequencies and prophage induction of the ΦSa3int phage Φ13. Our results show that the FQ resistance mutations did not affect ΦSa3int phage integration or prophage stability.

Strains and growth conditions
An overview of the strains used can be found in Table 1. S. aureus was grown and plated on tryptic soy broth or agar medium (TSB/TSA), containing 6 µg ml −1 ciprofloxacin (Cip) and/or 30 µg ml −1 kanamycin (Kan) if appropriate. E. coli was grown and plated on Luria-Bertani medium (LB) with 100 µg ml −1 ampicillin.

Strain constructions
The gyrA and grlA mutations conferring FQ resistance found in LA-MRSA CC398 were introduced in S. aureus 8325-4attB LA using pBASE6 [35] and via E. coli IM08B [36]. S. aureus 8325-4attB LA carries a 2 bp mutation in the bacterial attachment site in the hlb-gene, found in LA-S. aureus CC398 [11]. The genome sequence of S. aureus NCTC8325 (GenBank Accession no. NC_007795.1) was used as a template for construction of ca. 2 kb fragments containing the respective point mutations (grlA Ser80 → Tyr and gyrA Ser84 → Leu) with restriction sites at each end (purchased from Twist Bioscience) and amplified with primers and conditions stated in Table 2. The fragments were cloned into pBASE6 using BglII (AGATCT) and KpnI (GGTACC) and the resulting plasmids pBASE6:grlA and pBASE6:gyrA were purified using GeneJET Plasmid Mini Prep kit

Susceptibility testing
All 96 previously identified human LA-MRSA CC398 isolates from North Jutland, Denmark [13] were tested for resistance to norfloxacin by use of the disc diffusion method, in accordance with the European Committee on Antimicrobial Susceptibility Testing guidelines [40]. Minimal inhibitory concentration (MIC) for Cip was determined with microbroth dilution method [41]. Cip (Sigma Aldrich) was diluted to span a range between 256 and 0.5 µg ml −1 in the columns of a 96-well plate. Bacteria were added to the wells at a concentration of 1×10 5 c.f.u. ml −1 and MIC was determined as the lowest concentration without growth after overnight incubation at 37 °C.

Growth assessment
Doubling times were assessed by OD 600nm measurement in Bioscreen C MBR (Oy Growth Curves Ab Ltd). For this, 300 µl of the respective culture (starting OD 600nm =0.05) was added to each well followed by overnight incubation with shaking at 37 °C. OD 600nm was measured every 20 min. Doubling time was calculated by using the programme Growthrates 3.0 [42].

Lysogenization assay
Lysogenization with Φ13kan R was performed as described previously [11] and frequencies were calculated for 8325-4attB LA by dividing the number of colonies on TSA +Kan by the number of colonies on TSA and for 8325-4attB LA :FQ R by dividing the number of colonies on TSA +Kan+ Cip by the number of colonies on TSA +Cip.

Phage release
To determine phage release we selected lysogens with Φ13kan R in attB LA in the hlb gene. The cultures were freshly grown until OD 600nm =1 and either with or without the addition of 2 µg ml −1 Mitomycin C incubated for 2 h at 37 °C and 180 r.p.m. [11,12]. The cultures were centrifuged (5 min, 9000 r.p.m.), the supernatant sterile filtered using 0.2 µm membrane filters, 10-fold diluted in SM-buffer (100 mM NaCl, 50 mM Tris [pH=7.8], 1 mM MgSO 4 , 4 mM CaCl 2 ) and spotted on a lawn of S. aureus 8325-4. The lawn was prepared by adding 100 µl fresh culture (OD 600nm =1) to 3 ml top agar (mix of TSA (one part) and TSB (four parts) and 10 µM CaCl 2 ).

Statistical analysis
Statistical analysis was done in R

RESULTS AND DISCUSSION
While analysing a subset of LA-MRSA CC398 strains isolated from pigs (n=45) and humans (n=96) in Northern Jutland, Denmark [13], we noticed that all isolates harbouring a ΦSa3int prophage were also resistant to FQ and had mutations in gyrA/grlA ( Fig. 1 and Table 3). There was a significantly greater carriage of a ΦSa3int phage in strains containing at least one of these mutations than in the remaining strains (  . Spontaneous (grey bars) and mitomycin C induced phage release (black bars). Cultures were grown to OD 600nm =1 at which the starting cfu ml −1 was determined (black triangles). From then on, cultures were incubated for another 2 h either without or with the addition of 2 µg ml −1 mitomycin C.
To examine the impact of the grlA/gyrA mutations on ΦSa3int phage integration, we infected 8325-4attB LA and 8325-4attB LA :FQ R with a derivative of the ΦSa3int phage Φ13 that harbours a kanamycin resistance gene (Φ13kan R [11]) at a multiplicity of infection of one and monitored the number of kanamycin-resistant colonies arising (Fig. 2). We found no significant difference in the ability of Φ13kan R to integrate in the two strains and thus we conclude that the gyrA/grlA mutations do not affect the frequency with which ΦSa3int phages integrate. The slightly lower lysogenization frequency observed in S. aureus 8325-4attB LA :FQ R could be due to an increased doubling time of 41 min compared to 30 min for 8325-4attB LA (Fig. 3). However, the colony forming units on kanamycin-free medium were similar for wild-type and mutant cells under the conditions used for lysogenization (Table 4).
We further investigated whether the grlA/gyrA mutations influence spontaneous or mitomycin-induced prophage release from a lysogen. To this end, we monitored lysogens of 8325-4attB LA and 8325-4attB LA FQ R with Φ13kan R integrated as a prophage in attB LA . As shown in Fig. 4, no significant difference was found between the FQ-resistant or -sensitive isolates from three separate lysogens with three replicates each, both in regards to spontaneous or mitomycin C-induced phage release. Lysogen 8325-4attB LA FQ R 2 did not show spontaneous phage release, which may be due to the generally low number of released phages and the insensitivity of the assay. These data suggest that FQ resistant strains are not a favoured reservoir of ΦSa3int phages which otherwise could have explained why FQ resistant strains harbouring ΦSa3int phages were isolated from humans. However, it should be noted that Φ13 only shows up to 66 % sequence identity with the ΦSa3int phages found in the 17 CC398 strains. So even though the phages belong to the phage family with a Sa3 integrase [45] we cannot rule out that there may be phage specific factors encoded by the ΦSa3int phages of the CC398 strains which influence the phage integration/excision processes and are missing in Φ13. Detailed studies will be needed to examine this further.
In conclusion, with the ΦSa3int phage Φ13 and the S. aureus 8325-4attB LA model strain we did not observe an apparent link between FQ resistance and integration or stability of the phage. Future studies should be directed at unravelling the mechanisms leading to the frequent acquisition of IEC-carrying ΦSa3int phages by different LA-MRSA CC398 lineages to prevent further spread into the general population.

Funding information
This project has received funding from the European Union's Horizon 2020 research and innovation programme (grant no. 765147).

Author response to reviewers to Version 2
Dear Editor, We appreciate the constructive comments and have addressed the concerns and issues raised by the reviewers below,

Response to reviewer 1
The authors have studied the role of fluoroquinolone resistance mutations in grlA/gyrA on the integration of Sa3int into the genome of Staphylococcus aureus.
Sa3int phages are common lysogens of human associated lineages of S. aureus but are rarely found in livestock-associated genes. In this work the authors found a subset of strains, belonging to the CC398 clonal complex, that harbour a Sa13int phage. They found that these strains all carried mutations in grlA/gyrA, which render them resistant to fluoroquinolones.
The mutations in grlA/gyrA were constructed in a CC8 strain of S. aureus, which already had the CC398 attB site introduced to the hlb gene. They found that the mutations in grlA/gyrA had no impact on lysogeny of the prophage but that it did slow the growth rate of this strain. Similarly, they found that the mutations in grlA/gyrA had no impact on the frequency of phage excision and release.
Overall, this is a well-presented paper that explores an interesting question regarding the conditions required for Sa3int phage's to lysogenize in a CC398 strain. The experimental work is strong and I have added some suggestions regarding discussion of the literature. I believe a little restructuring and expansion of the introduction/conclusion will make the experimental work more understandable to a wider audience in the context of the literature.

Response:
We greatly appreciate the positive comments and have restructured the manuscript with expansion of introduction and discussion, which we believe have greatly improved the manuscript.

Major Revisions:
"Line 205-207 -The conclusions drawn are unclear. The authors hypothesised that the Sa3int phage could lysogenise in the CC398 lineage due to FQ resistant mutations. They have shown that these mutations have no effect on phage lysogeny or excision. They then state that this does not explain the distribution of FQ resistance in the pig population. However, the Sa3int phage is stated to be beneficial for infection of humans, not animals, and therefore it is unclear how the authors have come to this conclusion if Sa3int does not benefit infection in livestock.

Response:
We appreciate the comment and acknowledge that the conclusion was misplaced. Thus, the conclusion has been modified it as seen in lines 217-228.
The focus of this paper has been understanding how the Sa3int phage can integrate into CC398 strains. A discussion relating to alternative hypotheses and gaps in the knowledge would be beneficial to the readers understanding. When addressing this, authors should also discuss how this then links into the question of FQ resistance in the pig population. For example, the authors have not discussed the attB mutations and how these effect lysogeny" Response:We greatly appreciate this comment and acknowledge that the introduction and conclusion needed improvements. We have restructured the introduction with more focus on human isolates of CC398 strains (line 61-71) and we have modified the discussion to reflect how the FQ resistance could have been selected in humans had we seen enhanced carriage of Sa3int phages in these strains (lines 220). In terms of the attB mutations and how they affect lysogeny we have cited previous studies addressing this issue (lines 69-71).
" or the sequence diversity of the prophages lysogenized in CC398 strains in comparison to other Sa3int phages." Response:We compared the sequence between phi13 and the found prophages (up to 66% homology), as those are the phages we were interested in (added to discussion in lines 221-222). We find further comparisons with other Sa3int phages lie beyond the scope of this present study.

Minor Revisions:
"Line 65 -It is stated that the mutation in the attB site possibly reduces phage binding but has not been the focus of the study of this paper. The authors use 8325-4 with an attB mutated to have the sequence of the CC398 attB in the hlb gene. However, they do not assess if this has any effect on Sa3int phage integration/excision. Perhaps this will be the focus of another study or it has been tested previously, however, I feel like a greater discussion of the relevant literature is required on this topic, as it is possible that the attB sequence is not responsible for reduced Sa3int phage integration in CC398 strains."

Response:
We have cited previous studies where isogenic strains pairs carrying attB-wt and attB-LA were lysogenized with phi13 and where the mutations in LA strains reduced lysogenization and we highlight that we previously have observed integration in other locations on the chromosome in addition to the attB-LA site (Lines 69-71). Further, we find that the general restructuring of the introduction has improved the description of the background of the study and hope the reviewer agrees.
Line 66 -stated that spill over events are related to acquisition of IECs but in Fig. 1 the authors show that most of the lineage that is found to have the gyrA/grlA mutations are human isolates that do not harbor these elements. Greater clarity on livestock associated S. aureus and its association with humans/ human disease may help to clarify this.
Response: It has previously been seen that contact with livestock is a risk factor to human infections with LA strains while the presence of Sa3int phages increase the risk of permanent colonization and human-to-human colonization. We have included this information in line 60 and highlighted the importance for transmission even further in lines 72-76.
Line 102 -human isolates of S. aureus referenced again but most of the CC398 analysed are isolated from humans? (78% in the lineage studied). As above, expanding in the intro the role of LA infections in humans and how this links to FQ resistance and to IEC carriage may help to clarify this.

Response:
We apologize for this confusion and have now clarified this by stating non-livestock, human strains (line 68).
Line 69 -The authors have identified 20 CC398 strains in the same lineage that have a Sa3int prophage but only disclose FQ resistance as a difference. I understand that other difference in these strains may be the focus of further study and therefore cannot be disclosed, but if the authors can expand on the extent of the differences between the strains eg. Is FQ resistance the only consistent difference or are there other major differences such as insertions of large mobile elements etc.
Response:The strains have already been characterized in the study by Sieber et al 2020 (EID, reference #13), however, we added some details from this previous study to provide a better foundation for our study (lines 80-83).
The authors could also comment on the sequence homology of Sa3int phages that infect CC398 strains. Are these identical to Sa3int phages that infect other lineages of S. aureus or do they have distinct differences that may affect host range?
Response:We compared phi13 with the Sa3int phages from the CC398 strains and found sequence identity between 53 and 66%. Thus, we cannot rule out that there may be factors in the CC398 phages which could influence integration and/or excision that are not present in phi 13. However, both phi13 and the sampled phages employ a Sa3 integrase, which is considered mainly responsible for integration/excision, and therefore we find our findings valid. We have added this to the manuscript lines 222-226. Response to Reviewer 2 The manuscript "Fluoroquinolone resistance mediated by mutations in gyrA and grlA does not facilitate phage integration in Staphylococcus aureus" by Leinweber et al. characterises the impact of fluoroquinolone resistance mutations identified in livestock-associated (LA) Staphylococcus-aureus isolates on phage integration and release. Sa3 phages are rarely found in livestock, however a significantly larger proportion of mutants in gyrA/grlA were found to contain the Sa3 phage compared to non-resistant isolates. The authors reconstructed the relevant resistance mutations in a phage-cured lab strain that also had the modified attB site found in LA-isolates. Using a kanamycin-marked phi13 the authors then assessed the phage's ability to lysogenise into this host strain. The authors further look at the ability of the newly lysogenised, fluoroquinolone resistant strains to release phage progeny either spontaneously or after mitomycin C induction of the SOSO response.

General comments:
Overall, the manuscript is well written and easy to follow. However, I am less convinced by the generalised conclusions of the authors that there is no impact on lysogenisation or phage release. My main concern is that the authors used a lab phage (phi13) and not a phage derived from their isolates to assess the impact.
Response:We agree that further experiments with the phages stemming from our samples could be beneficial. However, we believe that phi13 is by now a well-studied model phage for this group and unfortunately isolating Sa3int phages has been rather difficult as the CC398 strains contain other prophages as well, so a more elaborate study would be necessary. Therefore, we believe that our data is of relevance despite this limitation.
Even though integration is facilitated into the Sa3 attachment site by the same integrase, this does not necessarily mean that the phages are the same and additional phage-encoded factors might also play an important role in the observed association of prophage and fluroquinolone resistance.
Response:We agree, and we have raised this issue in the discussion 222-227.
The authors should provide a comparison of the actual phage(s) found in the isolates and their model to support their hypothesis. Because the authors only test a single phage, they should modify their title and conclusions accordingly.
Response:We found a sequence homology between 53 and 66% and added this information as well as adjusted our title and conclusions in lines 219-227. I would also like to see a rational for the selection of an MOI of 1. At this MOI, almost 37% of the cells in the assay would never be infected with a phage. Could a higher MOI reveal a difference in terms of lysogenisation rates?
Response:Although not all phages may not be infected at this MOI we believe that it is already a high MOI compared to the MOI that may be found in natural surroundings wherever the phages may meet the LA-MRSA. With this in mind we believe that we have already tested a high MOI and have not seen a noticeable difference.

Specific comments:
Materials & Methods: Strain constructions are described incompletely and the counterselection steps against the temperature sensitive plasmid have not been described. Line 158: typo "incubatiion"

Response:We haveadjusted accordingly
Lines 192 -196: The authors state that there is a growth defect in the FQ-R strain compared to its sensitive parent (doubling time 41 min compared to 30 min, respectively). Is this a genuine growth defect of the mutant strain or the presence of the antibiotic causing this reduction? If it is not a genuine growth defect, why did the authors choose to maintain antibiotic selection throughout their experiments for a chromosomal mutant that should be stable? Wouldn't it be more comparable to use the same media for selection and determination of lysogens to rule out a confounding impact of growth conditions? Response:We agree completely with the reviewer on this point and redid the growth measurements in the absence of antibiotic. The data has been inserted as a new figure 3 and shows that there is practically no difference between the growth of the two FQ-R strain in the presence or absence of antibiotics.
When the authors state "However, the colony forming units on kanamycin-free medium were similar for wild type and mutant cells (Error! Reference source not found.) under the conditions used for lysogenization", does this refer to the reported colony numbers or did the authors perform an additional experiment omitting the antibiotic and observing identical CFU numbers?
Response:This number refers to the numbers reported here in the present manuscript.

Line 195: Reference missing
Response:The sentence is referring to the data in this manuscript, so no reference is needed. The referral to table 4 has been moved to the end of the sentence to highlight this further. -Are there additional prophages in these isolates?
Response:Yes, there are prophages in these isolates for example ΦSa1int, ΦSa3int, ΦSa7int, ΦSa9int and this information can be found in Sieber et al., 2020 (EID). As the focus of the present study was the IEC-carrying prophages we did not elaborate on the presence of other phages. Figure 2 and Table 4 should be combined as they represent the same data.
Response:We would prefer to keep both the table and the figure as the table shows that the actual number of cfu's in the respective experiments are equal, despite the difference in growth rate. However, if the editor or reviewer find this modification essential we will be happy to accommodate it.
It would be better to plot table 4 with individual datapoints for each replicate and provide statistical analysis of both the raw (table 4) and derived (figure2) data.

Response:
We have added average and standard deviation for the raw data. The derived data has been analysed with Mann-Whitney's t-test.
Also, it might make sense to plot the lysogenisation frequency on a logarithmic scale rather than a linear scale.

Response:
We considered this but believe that with data from a single time point the linear presentation is to be preferred. Figure 4: Why did the authors assess spontaneous and induced release after only 2 hours of incubation with or without mitomycin C. Do they have time resolved data showing that this is the best time-point to assess this? Many staphylococcal phages will release their progeny later than this and differences might become more evident after longer incubation. Also, the high OD at the start of the experiment might be detrimental for the induction process. the discussion to reflect how the FQ resistance could have been selected in humans had we seen enhanced carriage of Sa3int phages in these strains (lines 220). In terms of the attB mutations and how they affect lysogeny we have cited previous studies addressing this issue (lines 69-71).
Throughout -units throughout should be preceded by a space. Eg '100 mM' but most units have not followed this.
Response:We have corrected the grammatical and spelling errors.

Response to Reviewer 2
The manuscript "Fluoroquinolone resistance mediated by mutations in gyrA and grlA does not facilitate phage integration in Staphylococcus aureus" by Leinweber et al. characterises the impact of fluoroquinolone resistance mutations identified in livestock-associated (LA) Staphylococcus-aureus isolates on phage integration and release. Sa3 phages are rarely found in livestock, however a significantly larger proportion of mutants in gyrA/grlA were found to contain the Sa3 phage compared to non-resistant isolates. The authors reconstructed the relevant resistance mutations in a phage-cured lab strain that also had the modified attB site found in LA-isolates. Using a kanamycin-marked phi13 the authors then assessed the phage's ability to lysogenise into this host strain. The authors further look at the ability of the newly lysogenised, fluoroquinolone resistant strains to release phage progeny either spontaneously or after mitomycin C induction of the SOSO response.
General comments: Overall, the manuscript is well written and easy to follow. However, I am less convinced by the generalised conclusions of the authors that there is no impact on lysogenisation or phage release. My main concern is that the authors used a lab phage (phi13) and not a phage derived from their isolates to assess the impact.
Response:We agree that further experiments with the phages stemming from our samples could be beneficial. However, we believe that phi13 is by now a well-studied model phage for this group and unfortunately isolating Sa3int phages has been rather difficult as the CC398 strains contain other prophages as well, so a more elaborate study would be necessary. Therefore, we believe that our data is of relevance despite this limitation.
Even though integration is facilitated into the Sa3 attachment site by the same integrase, this does not necessarily mean that the phages are the same and additional phage-encoded factors might also play an important role in the observed association of prophage and fluroquinolone resistance.
Response:We agree, and we have raised this issue in the discussion 222-227.
The authors should provide a comparison of the actual phage(s) found in the isolates and their model to support their hypothesis. Because the authors only test a single phage, they should modify their title and conclusions accordingly.
Response:We found a sequence homology between 53 and 66% and added this information as well as adjusted our title and conclusions in lines 219-227.
I would also like to see a rational for the selection of an MOI of 1. At this MOI, almost 37% of the cells in the assay would never be infected with a phage. Could a higher MOI reveal a difference in terms of lysogenisation rates?
Response:Although not all phages may not be infected at this MOI we believe that it is already a high MOI compared to the MOI that may be found in natural surroundings wherever the phages may meet the LA-MRSA. With this in mind we believe that we have already tested a high MOI and have not seen a noticeable difference.

Specific comments:
Materials & Methods: Strain constructions are described incompletely and the counterselection steps against the temperature sensitive plasmid have not been described.

Response:
We have improved the description and have added in line 139-144.

Response:We haveadjusted accordingly
Lines 192 -196: The authors state that there is a growth defect in the FQ-R strain compared to its sensitive parent (doubling time 41 min compared to 30 min, respectively). Is this a genuine growth defect of the mutant strain or the presence of the antibiotic causing this reduction? If it is not a genuine growth defect, why did the authors choose to maintain antibiotic selection throughout their experiments for a chromosomal mutant that should be stable? Wouldn't it be more comparable to use the same media for selection and determination of lysogens to rule out a confounding impact of growth conditions?
Response:We agree completely with the reviewer on this point and redid the growth measurements in the absence of antibiotic. The data has been inserted as a new figure 3 and shows that there is practically no difference between the growth of the two FQ-R strain in the presence or absence of antibiotics.
When the authors state "However, the colony forming units on kanamycin-free medium were similar for wild type and mutant cells (Error! Reference source not found.) under the conditions used for lysogenization", does this refer to the reported colony numbers or did the authors perform an additional experiment omitting the antibiotic and observing identical CFU numbers?
Response:This number refers to the numbers reported here in the present manuscript.

Line 195: Reference missing
Response:The sentence is referring to the data in this manuscript, so no reference is needed. The referral to table 4 has been moved to the end of the sentence to highlight this further. -Are there additional prophages in these isolates?
Response:Yes, there are prophages in these isolates for example ΦSa1int, ΦSa3int, ΦSa7int, ΦSa9int and this information can be found in Sieber et al., 2020 (EID). As the focus of the present study was the IEC-carrying prophages we did not elaborate on the presence of other phages. Figure 2 and Table 4 should be combined as they represent the same data.
Response:We would prefer to keep both the table and the figure as the table shows that the actual number of cfu's in the respective experiments are equal, despite the difference in growth rate. However, if the editor or reviewer find this modification essential we will be happy to accommodate it.
It would be better to plot table 4 with individual datapoints for each replicate and provide statistical analysis of both the raw (table 4) and derived (figure2) data.

Response:
We have added average and standard deviation for the raw data. The derived data has been analysed with Mann-Whitney's t-test.
Also, it might make sense to plot the lysogenisation frequency on a logarithmic scale rather than a linear scale.

Response:
We considered this but believe that with data from a single time point the linear presentation is to be preferred. Figure 4: Why did the authors assess spontaneous and induced release after only 2 hours of incubation with or without mitomycin C. Do they have time resolved data showing that this is the best time-point to assess this? Many staphylococcal phages will release their progeny later than this and differences might become more evident after longer incubation. Also, the high OD at the start of the experiment might be detrimental for the induction process.

Response:
We have followed a protocol that we have used previously in our studies of Sa3int phages and to compared choose to follow this. We have cited Tang

Anonymous.
Date report received: 07 March 2023 Recommendation: Minor Amendment Comments: The manuscript "Fluoroquinolone resistance mediated by mutations in gyrA and grlA does not facilitate phage integration in Staphylococcus aureus" by Leinweber et al. characterises the impact of fluoroquinolone resistance mutations identified in livestock-associated (LA) Staphylococcus-aureus isolates on phage integration and release. Sa3 phages are rarely found in livestock, however a significantly larger proportion of mutants in gyrA/grlA were found to contain the Sa3 phage compared to non-resistant isolates. The authors reconstructed the relevant resistance mutations in a phage-cured lab strain that also had the modified attB site found in LA-isolates. Using a kanamycin-marked phi13 the authors then assessed the phage's ability to lysogenise into this host strain. The authors further look at the ability of the newly lysogenised, fluoroquinolone resistant strains to release phage progeny either spontaneously or after mitomycin C induction of the SOSO response. General comments: Overall, the manuscript is well written and easy to follow. However, I am less convinced by the generalised conclusions of the authors that there is no impact on lysogenisation or phage release. My main concern is that the authors used a lab phage (phi13) and not a phage derived from their isolates to assess the impact. Even though integration is facilitated into the Sa3 attachment site by the same integrase, this does not necessarily mean that the phages are the same and additional phage-encoded factors might also play an important role in the observed association of prophage and fluroquinolone resistance. The authors should provide a comparison of the actual phage(s) found in the isolates and their model to support their hypothesis. Because the authors only test a single phage, they should modify their title and conclusions accordingly. I would also like to see a rational for the selection of an MOI of 1. At this MOI, almost 37% of the cells in the assay would never be infected with a phage. Could a higher MOI reveal a difference in terms of lysogenisation rates? Specific comments: Materials & Methods: Strain constructions are described incompletely and the counterselection steps against the temperature sensitive plasmid have not been described. Line 158: typo "incubatiion" Lines 192 -196: The authors state that there is a growth defect in the FQ-R strain compared to its sensitive parent (doubling time 41 min compared to 30 min, respectively). Is this a genuine growth defect of the mutant strain or the presence of the antibiotic causing this reduction? If it is not a genuine growth defect, why did the authors choose to maintain antibiotic selection throughout their experiments for a chromosomal mutant that should be stable? Wouldn't it be more comparable to use the same media for selection and determination of lysogens to rule out a confounding impact of growth conditions? When the authors state "However, the colony forming units on kanamycin-free medium were similar for wild type and mutant cells (Error! Reference source not found.) under the conditions used for lysogenization", does this refer to the reported colony numbers or did the authors perform an additional experiment omitting the antibiotic and observing identical CFU numbers? Line 195: Reference missing Figure 1: -Numbers of strains in text and in figure do not match. In introduction (lines 69-71) referring to figure 1 state 20 isolates with Sa3 phage were resistant, however, in the results section (line 182) only 17/70 strains contained the prophage. Also, it is not quite clear how the numbers in Figure 1 (141 isolates) match to the sum of strains with or without phage in the results section (line 182: 26+70 = 96). -Are there additional prophages in these isolates? Figure 2 and Table 4 should be combined as they represent the same data. It would be better to plot table 4 with individual datapoints for each replicate and provide statistical analysis of both the raw (table 4) and derived (figure2) data. Also, it might make sense to plot the lysogenisation frequency on a logarithmic scale rather than a linear scale. Figure 4: Why did the authors assess spontaneous and induced release after only 2 hours of incubation with or without mitomycin C. Do they have time resolved data showing that this is the best time-point to assess this? Many staphylococcal phages will release their progeny later than this and differences might become more evident after longer incubation. Also, the high OD at the start of the experiment might be detrimental for the induction process.
Please rate the manuscript for methodological rigour Good